Culture microscope apparatus

ABSTRACT

A culture microscope apparatus has an illumination unit to apply excitation light to the specimen, a specimen observing portion to observe light generated from the specimen due to the excitation light, and a dimmer unit to dim the excitation light that has penetrated the specimen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority fromprior Japanese Patent Applications No. 2003-365025, filed Oct. 24, 2003;and No. 2004-142635, filed May 12, 2004, the entire contents of both ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a culture microscope apparatus toobserve living cells that have been kept alive under a constantenvironmental condition for long-term observation of cells of livingorganisms such as animals or plants.

2. Description of the Related Art

Generally, in such fields as biochemistry, living cells of animals orplants are kept alive under a suitable condition to observe behavior ofthe living cells for functional analysis of living organisms.

For the behavioral observation of living cells, fluorescent dyes usingantigen antibody reactions and fluorescent observation usinggene-transferred fluorescent protein are utilized. The fluorescentobservation is used because it enables observation on the molecularlevel and behavioral observation of distributions and molecules. It isto be noted here that the fluorescence is a phenomenon in which ifenergy such as ultraviolet rays is applied to a substance from theoutside, atoms of the substance transit from ground state to excitedstate and then emit specific light when returning to the ground state.The ultraviolet rays from the outside are generally called excitationlight.

On the other hand, there is a phenomenon called discoloration in whichif the fluorescent dyes and the fluorescent protein are continuouslyexposed to the excitation light, the intensity of fluorescence light isgradually lowered or nullified. Therefore, the fluorescent observationrequires attention, for example, using as weak excitation light aspossible and exposing a sample to the excitation light duringobservation only.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed, according to an aspect of theinvention, to a culture microscope apparatus that reduces discolorationof fluorescent dyes. The culture microscope apparatus according to thepresent invention comprises illumination means for applying excitationlight to a specimen, specimen observing means for observing lightgenerated from the specimen due to the excitation light, and dimmermeans for dimming the excitation light that has penetrated the specimen.

The present invention is directed, according to another aspect of theinvention, to a culture microscope apparatus that has less temperaturechanges in an installation environment and is easily cleaned. Theculture microscope apparatus according to the present inventioncomprises a microscope device to observe a specimen, a culture devicecapable of controlling temperature and humidity, and isolation means forisolating the microscope device from moisture of the culture device.

Advantages of the invention will be set forth in the description whichfollows, and in part will be obvious from the description, or may belearned by practice of the invention. Advantages of the invention may berealized and obtained by means of the instrumentalities and combinationsparticularly pointed out hereinafter.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate embodiments of the invention, andtogether with the general description given above and the detaileddescription of the embodiments given below, serve to explain theprinciples of the invention.

FIG. 1 shows a schematic configuration of a first embodiment of thepresent invention;

FIG. 2 shows a schematic configuration of essential parts of the firstembodiment;

FIG. 3 shows a schematic configuration of essential parts of a secondembodiment of the present invention;

FIG. 4 shows a schematic configuration of essential parts of a thirdembodiment of the present invention;

FIG. 5 shows a schematic configuration of an externally installed partused for the third embodiment;

FIG. 6 shows a schematic configuration of essential parts of a fourthembodiment of the present invention;

FIG. 7 shows a schematic configuration of a conventional culturemicroscope apparatus;

FIG. 8 schematically shows a culture microscope apparatus of a fifthembodiment of the present invention;

FIG. 9 shows in an enlarged manner a peripheral part of a table shown inFIG. 8;

FIG. 10 shows in an enlarged manner a peripheral part of an objectivelens shown in FIG. 8;

FIG. 11 shows an illumination device applicable to a microscope deviceshown in FIG. 8;

FIG. 12 shows another illumination device applicable to the microscopedevice shown in FIG. 8;

FIG. 13 shows a modification of the culture microscope apparatus of thefifth embodiment of the present invention;

FIG. 14 shows another modification of the culture microscope apparatusof the fifth embodiment of the present invention;

FIG. 15 shows still another modification of the culture microscopeapparatus of the fifth embodiment of the present invention;

FIG. 16 shows further still another modification of the culturemicroscope apparatus of the fifth embodiment of the present invention;and

FIG. 17 schematically shows the culture microscope apparatus of a sixthembodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION First Embodiment

FIG. 7 shows a schematic configuration of a conventional culturemicroscope apparatus disclosed in Jpn. Pat. Appln. KOKAI Publication No.2003-93041. In this culture microscope apparatus, living samplescontained in culture cases 1503 placed on sample tables 1502 in aculture container 1501 are cultured in the culture container 1501, andimages of the living samples are picked up by a CCD 1504 forobservation. In order to observe fluorescent images of the livingsamples, light from a fluorescence measurement excitation light source1505 is projected from an excitation light projection fiber 1506,reflected by mirrors 1507 and 1508, and applied as excitation lightthrough objective lens 1509 to the living sample in the culture case1503, and then an image of fluorescence light emitted from the livingsample is picked up by the CCD 1504 through the objective lens 1509. Onthe other hand, for observation of transmitted light image of the livingsample, light from a white light source 1510 is applied to the livingsample in the culture case 1503 through a white light projection fiber1511, and an image of light that has penetrated the living sample ispicked up by the CCD 1504 through the objective lens 1509.

In the culture microscope apparatus of Jpn. Pat. Appln. KOKAIPublication No. 2003-93041, most of the excitation light that has beenprojected from the excitation light projection fiber 1506 and appliedthrough the objective lens 1509 to the living sample in the culture case1503 may penetrate the culture case 1503 and be reflected by the surfaceof a fixed arm 1512 supporting an projection end of the fiber 1511, sothat the reflected excitation light might excite the living samples thatare not targeted for observation to fade fluorescent dyes. Further, evenif all the excitation light that has penetrated the culture case 1503can be captured by the white light projection fiber 1511, lightreflected by a lamp of the white light source 1510 or the like mayreturn through the white light projection fiber 1511 to fade thefluorescent dyes.

In view of such circumstances, the present embodiment is directed to aculture microscope apparatus that reduces the discoloration of thefluorescent dyes.

FIG. 1 shows a schematic configuration of the culture microscopeapparatus to which the present invention is applied, and FIG. 2 shows anenlarged schematic configuration of essential parts of the culturemicroscope apparatus.

A culture microscope apparatus body 1101 is provided with a culturecontainer 1102. The culture container 1102 is provided with multistage(three stages in an example shown) culture case support racks 1104. Eachof the culture case support racks 1104 supports (three in an exampleshown) specimens (i.e., culture cases 1103 containing living cells1129). The culture case support rack 1104 is provided with a recess 1104a slightly larger than the culture case 1103, and an opening 1104 bslightly smaller than the culture case 1103 on a bottom surface of therecess 1104 a, as shown in FIG. 2. Thus, the culture case support rack1104 uses the recess 1104 a to position the culture case 1103 and allowsof observation of the living cells 1129 in the culture case 1103 throughthe opening 1104 b.

Here, the culture case 1103 comprises, for example, a dish and a microwell-plate to which a culture solution is added, and a necessary amountof living cells 1129 is distributed and contained in the culture case1103. Further, the culture case 1103 is made of a resin material towhich a colorless and transparent glass is affixed or in which a glassis affixed on its bottom surface.

A valve 1135 is connected to the culture container 1102. This valve 1135is for supplying carbon dioxide into the culture container 1102. Atemperature adjustment heater 1137 for temperature adjustment and anevaporating dish 1136 retaining water are located on the bottom surfaceof the culture container 1102, and a humidification heater 1140 islocated under the evaporating dish 1136. Further, a sensor 1141 isprovided in the culture container 1102. This sensor 1141 is intended todetect carbon dioxide concentration, humidity and temperature in theculture container 1102.

A controller 1138 is connected to the valve 1135, the temperatureadjustment heater 1137, the humidification heater 1140 and the sensor1141. The controller 1138 is intended to perform various kinds ofcontrol; for example, if the carbon dioxide concentration, humidity andtemperature in the culture container 1102 are detected by the sensor1141, the controller 1138 opens the valve 1135 in accordance with anoutput of the detection to supply carbon dioxide, turns on thehumidification heater 1140 to evaporate water from the evaporating dish1136 for supply of steam, or turns on the temperature adjustment heater1137 to supply heat for adjustment of temperature in the culturecontainer 1102. This maintains a constant environmental condition in theculture container 1102 necessary for the living cells 1129 so that theliving cells 1129 can stay alive for a long period of time in theculture container 1102.

On the other hand, a pair of first guides 1105 and a first movementscrew 1106 are arranged on the bottom surface of the culture container1102 in parallel in a direction perpendicular to the drawing.

The first guide 1105 has a fixed portion 1105 a and a moving portion1105 b, and the moving portion 1105 b is linearly movable in contrastwith the fixed portion 1105 a. The first movement screw 1106 has a screwportion 1106 a and a nut portion 1106 b, and the screw portion 1106 a isturned while the rotation of the nut portion 1106 b is regulated so thatthe nut portion 1106 b moves straight.

A first moving part 1107 is attached to the moving portion 1105 b of thefirst guide 1105 and the nut portion 1106 b of the first movement screw1106. Further, a first drive portion 1109 is attached to the screwportion 1106 a of the first movement screw 1106. The first drive portion1109 rotationally drives the screw portion 1106 a of the first movementscrew 1106 under instructions from the controller 1138, and the rotationof the screw portion 1106 a is converted to straight movement of the nutportion 1106 b, thereby enabling the first moving part 1107 to move in adirection (direction perpendicular to the drawing) guided by the firstguide 1105.

In the first moving part 1107, a pair of guide mounting surfaces 1107 a,1107 b is provided to face each other perpendicularly to the bottomsurface of the culture container 1102. The guide mounting surface 1107 ais provided with a second guide 1110, and the guide mounting surface1107 b is provided with a second movement screw 1108. The second guide1110 and the second movement screw 1108 are configured in the samemanner as the first guide 1105 and the first movement screw 1106. Inother words, the second guide 1110 has a fixed portion 1110 a fixed tothe guide mounting surface 1107 a and a moving portion 1110 b, andallows the moving portion 1110 b to move linearly relative to the fixedportion 1110 a. Further, the second movement screw 1108 has a screwportion 1108 a fixed to the guide mounting surface 1107 a and a nutportion 1108 b, and the screw portion 1108 a is turned so that the nutportion 1108 b moves straight.

A second moving part 1111 is attached to the moving portion 1110 b ofthe second guide 1110 and the nut portion 1108 b of the second movementscrew 1108. Further, a second drive portion 1139 is attached to thescrew portion 1108 a of the second movement screw 1108. The second driveportion 1139 rotationally drives the screw portion 1108 a of the secondmovement screw 1108 under instructions from the controller 1138, and therotation of the screw portion 1108 a is converted to straight movementof the nut portion 1108 b, thereby enabling the second moving part 1111to move in a direction (vertical direction of the drawing) guided by thesecond guide 1110.

The second moving part 1111 is made from a rectangular frame, and haslongitudinal portions 1111 a, 1111 b along the guide mounting surfaces1107 a, 1107 b and guide support portions 1111 c, 1111 d parallel withthe culture case support rack 1104.

The guide support portion 1111 c is provided with a third guide 1113 anda third movement screw 1114, and the guide support portion 1111 d isprovided with a fourth guide 1115 and a fourth movement screw 1116. Thethird guide 1113 and the fourth guide 1115 are configured in the samemanner as the first guide 1105. The third movement screw 1114 and thefourth movement screw 1116 are also configured in the same manner as thefirst movement screw 1106. Here, the third movement screw 1114 has ascrew portion 1114 a fixed to the guide support portion 1111 c and a nutportion 1114 b, and the screw portion 1114 a is turned so that the nutportion 1114 b moves straight. The fourth movement screw 1116 also has ascrew portion 1116 a to the guide support portion 1111 d and a nutportion 1116 b, and the screw portion 1116 a is turned so that the nutportion 1116 b moves straight.

Specimen observing means or a specimen observing portion 1121 isattached to the nut portion 1114 b of the third movement screw 1114.Further, a third drive portion 1117 is attached to the screw portion1114 a of the third movement screw 1114. The third drive portion 1117rotationally drives the screw portion 1114 a of the third movement screw1114 under instructions from the controller 1138, and the rotation ofthe screw portion 1114 a is converted to straight movement of the nutportion 1114 b, thereby enabling the specimen observing portion 1121 tomove in accordance with the third guide 1113 in a direction (horizontaldirection of the drawing) along the culture case support rack 1104.

Dimmer means or a dimmer unit 1122 is attached to the nut portion 1116 bof the fourth movement screw 1116. Further, a fourth drive portion 1118is attached to the screw portion 1116 a of the fourth movement screw1116. The fourth drive portion 1118 rotationally drives the screwportion 1116 a of the fourth movement screw 1116 under instructions fromthe controller 1138, and the rotation of the screw portion 1116 a isconverted to straight movement of the nut portion 1116 b, therebyenabling the dimmer means or dimmer unit 1122 to move in accordance withthe fourth guide 1115 in a direction (horizontal direction of thedrawing) along the culture case support rack 1104.

In this case, the controller 1138 simultaneously drives the thirdmovement screw 1114 by the third drive portion 1117 and the fourthmovement screw 1116 by the fourth drive portion 1118, and controls sothat the specimen observing portion 1121 faces the dimmer means ordimmer unit 1122 with the culture case 1103 put between them and anoptical axis X of the specimen observing portion 1121 coincides with anoptical axis Y of the dimmer means or dimmer unit 1122.

The specimen observing portion 1121 is connected with Illumination meansor an illumination unit. The illumination means or illumination unitincludes an incident-light fiber 1124 and an external illuminationportion 1123. The specimen observing portion 1121 is connected to theexternal illumination portion 1123 through the incident-light fiber1124. The external illumination portion 1123 comprises a mercury lamp, axenon lamp, a laser light source or the like.

The specimen observing portion 1121 has, as an observation opticalsystem to observe the living cells 1129 in the culture case 1103, afirst lens 1134, an excitation filter 1125, an objective lens 1131, adichroic mirror 1127, an emission filter 1126 and an image-forming lens1144, and also has image pickup means or an image pickup device 1128such as a CCD. Here, the excitation filter 1125 has properties that onlytransmit wavelengths necessary for the excitation of the fluorescentdyes. The dichroic mirror 1127 has properties that reflect theexcitation light having a relatively short wavelength and transmit therelatively fluorescence light having a long wavelength. Moreover, theemission filter 1126 has properties that selectively transmit thefluorescence light emitted by the fluorescent dyes.

Furthermore, if illumination light from the external illuminationportion 1123 is introduced to the specimen observing portion 1121through the incident-light fiber 1124, the light projected from theincident-light fiber 1124 will be light converging on a rear focal planeof the objective lens 1131 through the first lens 1134, and the light isselected by the excitation filter 1125, reflected by the dichroic mirror1127 and converges on the rear focal plane of the objective lens 1131.Further, the light converging on the rear focal plane of the objectivelens 1131 is brought into parallel light by the objective lens 1131 toilluminate an illumination range uniformly as excitation light 1142 thatexcites the fluorescent dyes in the living cells 1129 in the culturecase 1103. The fluorescent dyes excited by the excitation light 1142emit fluorescence light 1143 having a wavelength longer than that of theexcitation light 1142. The fluorescence light 1143 is brought intoparallel light by the objective lens 1131, penetrates the dichroicmirror 1127 and the emission filter 1126, and falls on an image pickupplane of the image pickup device 1128 through the image-forming lens1144, thereby picking up an image thereof.

Meanwhile, the illumination range is dotted with the living cells 1129,and parts to be observed within the living cells 1129 are furtherlimited and less. Thus, most of the excitation light 1142 in theillumination range penetrates the culture case 1103 to arrive at thedimmer means or dimmer unit 1122.

The dimmer means or dimmer unit 1122 has a sealing glass 1132, areflective member 1122 a and a light absorption member 1122 b to dim theexcitation light penetrating the culture case 1103. In this case, thesealing glass 1132 is installed obliquely with respect to the opticalaxis X of the specimen observing portion 1121 so that the excitationlight 1142 penetrating the culture case 1103 is reflected by the surfaceof the sealing glass 1132 and the reflected light will not return to theside of the living cells 1129. The reflective member 1122 a is locatedwith a normal at 45 degrees to the optical axis Y so that the excitationlight 1142 penetrating the sealing glass 1132 is reflectedperpendicularly to the optical axis Y. The light absorption member 1122b is located on a reflected light path of the reflective member 1122 a.For example, a hair transplant paper 1133 is attached to the lightabsorption member 1122 b to absorb the excitation light.

In such a configuration, the first moving part 1107 is moved back andforth along the first guide 1105 by the first movement screw 1106 andthe second moving part 1111 is moved up and down along the second guide1110 by the second movement screw 1108 in order to set the heightpositions of the specimen observing portion 1121 and the dimmer means ordimmer unit 1122 relative to the respective culture case support racks1104. Moreover, the third movement screw 1114 and the fourth movementscrew 1116 are driven so that the specimen observing portion 1121 andthe dimmer means or dimmer unit 1122 move along the culture case supportracks 1104 in order to position the specimen observing portion 1121 andthe dimmer means or dimmer unit 1122 above the living cells 1129 in theculture case 1103 on the culture case support rack 1104. In this case,the specimen observing portion 1121 and the dimmer means or dimmer unit1122 are set in such a state that the optical axis X of the specimenobserving portion 1121 coincides with the optical axis Y of the dimmermeans or dimmer unit 1122 so that the living cells 1129 in the culturecase 1103 are placed between them.

In this state, if the illumination light from the external illuminationportion 1123 is introduced to the specimen observing portion 1121through the incident-light fiber 1124, the light projected from theincident-light fiber 1124 will be light converging on the rear focalplane of the objective lens 1131 through the first lens 1134, and thelight is selected by the excitation filter 1125, reflected by thedichroic mirror 1127 and converges on the rear focal plane of theobjective lens 1131. The light converging on the rear focal plane of theobjective lens 1131 is brought into parallel light by the objective lens1131 to illuminate the illumination range uniformly as excitation light1142 that excites the fluorescent dyes in the living cells 1129 in theculture case 1103. The fluorescent dyes excited by the excitation light1142 emit the fluorescence light 1143 having a wavelength longer thanthat of the excitation light 1142. The fluorescence light 1143 isbrought into the parallel light by the objective lens 1131, penetratesthe dichroic mirror 1127 and the emission filter 1126, and falls on theimage pickup plane of the image pickup device 1128 through theimage-forming lens 1144, thereby picking up an image thereof.

On the other hand, the excitation light 1142 that penetrates the culturecase 1103 and that is not used for excitation reaches the dimmer meansor dimmer unit 1122 and falls on the sealing glass 1132. In this case,as the sealing glass 1132 is provided obliquely with respect to theoptical axis X of the specimen observing portion 1121, a slight amountof reflection on the surface of the sealing glass 1132 returns to theobserved parts of the living cells 1129 without causing unevenillumination. Further, the excitation light 1142 that has penetrated thesealing glass 1132 is reflected by the reflective member 1122 a, reachesthe light absorption member 1122 b and is absorbed by the hairtransplant paper 1133.

Therefore, the excitation light 1142 that has penetrated the culturecase 1103 and that is not used for excitation is led to and dimmed bythe dimmer means or dimmer unit 1122 without returning to the side ofthe living cells 1129, thereby making it possible to significantlyreduce the discoloration of the fluorescent dyes in the living cells1129 that are not targeted for observation. This allows the fluorescentimage with good contrast to be observed for a long period of time.

Furthermore, since the hair transplant paper 1133 used for the dimmermeans or dimmer unit 1122 is sealed in the dimmer means or dimmer unit1122 by the sealing glass 1132, it is also possible to preventcontamination inside the culture container 1102.

Second Embodiment

Next, a second embodiment of the present invention will be described.The present embodiment is directed to another dimmer unit applicableinstead of the dimmer unit of the first embodiment. Therefore, as theculture microscope apparatus to which the present embodiment is appliedis similar to that described with reference to FIG. 1, FIG. 1 isincorporated herein.

FIG. 3 shows a schematic configuration of essential parts of the culturemicroscope apparatus of the present invention. In FIG. 3, the samenumerals are given to the same parts as those in FIG. 2.

Dimmer means or a dimmer unit 1201 in the present embodiment has asecond lens 1202, a pin hole 1203 and a scattering portion 1204. The pinhole 1203 is set greater than the diameter of the excitation light 1142converged by the second lens 1202. The scattering portion 1204 absorbsand scatters the incident excitation light 1142 and has low reflectingcoating inside.

In such a configuration, the excitation light 1142 penetrating theculture case 1103 is converged into the pin hole 1203 by the second lens1202. The light that has passed through the pin hole 1203 enters thescattering portion 1204. The scattering portion 1204 absorbs andscatters the incident excitation light 1142 with the low reflectingcoating therein.

Thus, the excitation light 1142 is repeatedly absorbed and scattered inthe scattering portion 1204 such that it is progressively dimmed toreduce energy. In this case, the excitation light repeatedly reaches thepin hole 1203 several times, thereby making it possible to sufficientlyreduce damage to the living cells 1129 even if part of the light returnsto the living cells 1129.

Again, in this case, if the above-mentioned hair transplant paper 1133is provided in the scattering portion 1204 and the sealing glass 1132described in detail is provided in the pin hole 1203, the degree ofdimming can be increased while the contamination of the living cells1129 is reduced.

Third Embodiment

Next, a third embodiment of the present invention will be described. Thepresent embodiment is directed to another dimmer unit applicable insteadof the dimmer unit of the first embodiment. Therefore, as the culturemicroscope apparatus to which the present embodiment is applied issimilar to that described with reference to FIG. 1, FIG. 1 isincorporated herein.

FIG. 4 and FIG. 5 show schematic configurations of essential parts ofthe culture microscope apparatus of the present invention. In FIG. 4 andFIG. 5, the same numerals are given to the same parts as those in FIG.2.

Dimmer means or a dimmer unit 1301 in the present embodiment has a thirdlens 1302 and a transmitted-light fiber 1303, and further has anexternally installed part 1304 attached to the culture microscopeapparatus body 1101. The third lens 1302 converges the excitation light1142 that has penetrated the living cells 1129 to an end of thetransmitted-light fiber 1303. The transmitted-light fiber 1303 transmitsthe excitation light 1142 introduced through the third lens 1302 to theexternally installed part 1304.

In the externally installed part 1304, a lens 1302 a and an illuminationlight source 1305 are located on an optical axis V at an end of thetransmitted-light fiber 1303, as shown in FIG. 5. A dimmer portion 1306is located on an optical axis W vertical to the optical axis V at theend of the transmitted-light fiber 1303. A reflecting mirror 1307 islocated at an intersecting point Z of the optical axis V and the opticalaxis W. The reflecting mirror 1307 is located to form an angle of 45° tothe optical axes V, W and is movable in a direction of the optical axisW.

The reflecting mirror 1307 is provided with a fifth guide 1310 and afifth movement screw 1311. The fifth guide 1310 has a fixed portion 1310a fixed to the side of the externally installed part 1304 and a movingportion 1310 b, and allows the moving portion 1310 b to move linearlyrelative to the fixed portion 1310 a. Further, the fifth movement screw1311 has a screw portion 1311 a fixed, in a manner to be able to turn,to the side of the externally installed part 1304 and a nut portion 1311b, and the screw portion 1311 a is turned so that the nut portion 1311 bmoves straight.

The reflecting mirror 1307 is attached to the moving portion 1310 b ofthe fifth guide 1310 and the nut portion 1311 b of the fifth movementscrew 1311. Further, a fifth drive portion 1312 is attached to the screwportion 1311 a of the fifth movement screw 1311. The fifth drive portion1312 rotationally drives the screw portion 1311 a of the fifth movementscrew 1311 under instructions from the controller 1138, and the rotationof the screw portion 1311 a is converted to straight movement of the nutportion 1311 b, thereby enabling the reflecting mirror 1307 to move in adirection (direction of the optical axis W) guided by the fifth guide1310.

Position sensors 1308 a, 1308 b are located on a travel path of thereflecting mirror 1307. The position sensor 1308 a detects that thereflecting mirror 1307 is located at the intersecting point Z of theoptical axis V and the optical axis W, and the position sensor 1308 bdetects that the reflecting mirror 1307 is located, on the optical axisW, at an evacuated position where it does not prevent the excitationlight projected from the transmitted-light fiber 1303. Thereby, thecontroller 1138 can control the position of the reflecting mirror 1307by use of the detected positions of the position sensors 1308 a, 1308 b,so that an optical connection end of the transmitted-light fiber 1303can be switched between the dimmer portion 1306 and the illuminationlight source 1305.

According to such a configuration, when the reflecting mirror 1307 islocated at the intersecting point Z, the excitation light 1142 projectedfrom the transmitted-light fiber 1303 is brought into parallel light bythe lens 1302 a, and is reflected by the reflecting mirror 1307 andreaches the dimmer portion 1306. In this case, again, if theabove-mentioned hair transplant paper 1133 is provided in the dimmerportion 1306, the excitation light 1142 is absorbed and dimmed by thehair transplant paper 1133.

On the other hand, when the reflecting mirror 1307 is moved to theevacuated position on the optical axis W where it does not prevent theexcitation light projected from the transmitted-light fiber 1303, thetransmitted-light fiber 1303 is optically connected to the illuminationlight source 1305, so that the light from the illumination light source1305 is projected from the third lens 1302 through the transmitted-lightfiber 1303, and is applied to the living cells 1129 from the oppositeside of the specimen observing portion 1121 with reference to the livingcells 1129.

Thus, the position of the reflecting mirror 1307 can be switched to leadthe excitation light 1142 that has penetrated the culture case 1103 tothe side of the dimmer portion 1306 for dimming in the case ofincident-light illumination so that the discoloration of the livingcells 1129 can be prevented. On the one hand, if the light from theillumination light source 1305 illuminates the living cells 1129 at thetime of transmitted-light illumination, a transmitted image of theliving cells 1129 can be observed.

Fourth Embodiment

Next, a fourth embodiment of the present invention will be described.The present embodiment is directed to another dimmer unit applicableinstead of the dimmer unit of the first embodiment. Therefore, as theculture microscope apparatus to which the present embodiment is appliedis similar to that described with reference to FIG. 1, FIG. 1 isincorporated herein.

FIG. 6 shows a schematic configuration of essential parts of the culturemicroscope apparatus of the present invention. In FIG. 6, the samenumerals are given to the same parts as those in FIG. 3.

The culture microscope apparatus of the present embodiment comprises atransmitted-light fiber 1401, a fourth lens 1402 and an externallyprovided illumination light source 1403, in addition to the dimmer meansor dimmer unit 1201 described in the second embodiment. The illuminationlight source 1403 is optically connected to the fourth lens 1402 throughthe transmitted-light fiber 1401, and light from the illumination lightsource 1403 is projected from the fourth lens 1402 through thetransmitted-light fiber 1401.

According to such a configuration, if the optical axis Y of the dimmermeans or dimmer unit 1201 is adapted to coincide with the optical axis Xof the specimen observing portion 1121, the excitation light 1142 thathas penetrated the culture case 1103 is introduced into the dimmer meansor dimmer unit 1201 and dimmed.

Furthermore, if the dimmer means or dimmer unit 1201 is moved by thefourth guide 1115, the fourth movement screw 1116 and the fourth driveportion 1118, and an optical axis T on the side of the fourth lens 1402and the transmitted-light fiber 1401 is adapted to coincide with theoptical axis X of the specimen observing portion 1121, the light fromthe illumination light source 1403 is projected from the fourth lens1402 through the transmitted-light fiber 1401, and is applied to theliving cells 1129 from the opposite side of the specimen observingportion 1121 with reference to the living cells 1129. This enables theliving cells 1129 to be illuminated with the light from the illuminationlight source 1403 to obtain a transmission observation image.

In this way, the excitation light 1142 can be dimmed and observation ofthe living cells 1129 with transmitted illumination can be performedwithout specially providing means or a mechanism to switch the positionof the reflecting mirror 1307 described in the third embodiment.

Fifth Embodiment

The background of the present embodiment will first be described.

Because living organisms have a high degree of complexity, it is noteasy to understand their structures and functions. Therefore, a simpleexperiment system has recently been utilized, and it uses cells that areminimum units capable of reproducing a life phenomenon, that is, thecultured cells. The cultured cells are used to enable an experiment inwhich analyses of a hormone response and the like are not affected byother factors in the living organism.

In other words, functional analysis of genes can be implemented by theintroduction and inhibition of genes. It is necessary to use anenvironment simulating the inside of the living organism in order toculture cells. Therefore, the temperature is set at a body temperatureof 37° C., and a culture medium simulating intercellular fluid is used.The culture medium includes a carbonic acid buffer for PH adjustment inaddition to sources of nutrition such as amino acid. The carbonic acidbuffer is in an equilibrium state in the presence of air containingcarbon dioxide gas at a high partial pressure of 5%, and used forculture in an open system such as a dish. Moreover, a highly humidenvironment is required to prevent water from evaporating from theculture medium.

To culture cells, a carbon dioxide gas incubator that has theabove-mentioned environmental conditions is used.

A phase contrast microscope is used to observe the state of cells and afluorescence microscope is used to observe the expression of GFP,thereby performing time-lapse observation based on time-series imageacquirement.

However, the microscope is generally placed outside the carbon dioxidegas incubator, the observation causes changes in, for example,temperature and PH to cells to give stress to the cells, which mightaffect an experimental result.

Therefore, Jpn. Pat. Appln. KOKAI Publication No. 2003-93041 proposes anapparatus in which a movable device and a microscope are arranged in anincubator capable of controlling carbon dioxide gas, temperature andhumidity. This apparatus makes it possible to observe cells in a cultureenvironment without taking the cultured cells from the cultureenvironment.

Furthermore, Jpn. Pat. Appln. KOKAI Publication No. 10-28576 proposes amicroscopic observation transparent constant temperature cultureapparatus that is installed on a microscope and that can control carbondioxide gas, temperature and humidity.

If the cultured cells are contaminated with microorganisms such asbacteria and molds having a high proliferation rate, the cultured cellsrun out of nutrients and die. Culture instruments are disinfected andsterilized for prevention of contamination, but the apparatus of Jpn.Pat. Appln. KOKAI Publication No. 2003-93041 comprises moving means or amoving mechanism in the culture apparatus, thus having a complicatedshape in the apparatus and having difficulty in cleaning. This will be afactor that restricts the sufficient prevention of contamination.

Moreover, in the apparatus of Jpn. Pat. Appln. KOKAI Publication No.10-28576, a container is constructed in a limited space on themicroscope, so that temperature setting is easily changed by theatmospheric environment.

In view of such circumstances, the present embodiment is directed to theculture microscope apparatus that causes a small change in an installedenvironment and that is easily cleaned.

FIG. 8 schematically shows the culture microscope apparatus of a fifthembodiment of the present invention. The culture microscope apparatusbasically comprises a microscope device to observe cultured cells and aculture device 2101 capable of controlling temperature, humidity andcarbon dioxide gas concentration so that they have values suitable forthe cultured cells.

[Outline of Microscope Device]

In FIG. 8, the microscope device comprises an objective lens 2125, whichallows of observation of the cultured cells in a specimen 2123 (i.e.,culture case containing the cultured cells), an image pickup device2150, which picks up an image of the cultured cells enlarged by theobjective lens 2125, a moving device, which relatively moves thespecimen 2123 (i.e., the cultured cells) and the objective lens 2125, anupper base member 2114 on which the objective lens 2125 and the movingdevice are placed, a lower base member 2191 on which the image pickupdevice 2150 is placed, and support columns 2111 coupling the upper basemember 2114 to the lower base member 2191.

The upper base member 2114, the lower base member 2191 and the supportcolumns 2111 are all made of a low expansion material with a littleexpansion due to heat.

The moving device comprises a straight moving guide 2115, a horizontallymoving member 2116, a ball screw 2117, a stepping motor 2118, a rotationshaft 2119, a rotation shaft bearing 2120, a stepping motor 2121, atable 2122, a straight moving guide 2124, a vertically moving member2126, a ball screw 2127 and a stepping motor 2128.

The straight moving guide 2115, the horizontally moving member 2116, theball screw 2117 and the stepping motor 2118 are all provided under theupper base member 2114. The straight moving guide 2115 supports thehorizontally moving member 2116 movably in one direction, and thestepping motor 2118 moves the horizontally moving member 2116 throughthe ball screw 2117. The horizontally moving member 2116 holds therotation shaft bearing 2120 and the stepping motor 2121. The steppingmotor 2121 is located coaxially with the rotation shaft bearing 2120.The rotation shaft bearing 2120 supports the rotation shaft 2119rotatably vertically to a horizontal plane and pressurizes the rotationshaft 2119. Balls 2135 are provided between the rotation shaft 2119 andthe rotation shaft bearing 2120 to reduce friction (see FIG. 9). A lowerend of the rotation shaft 2119 is connected to the stepping motor 2121.An upper end of the rotation shaft 2119 protrudes from an upper surfaceof the upper base member 2114 through an opening formed in the upperbase member 2114. The table 2122 on which the specimen 2123 is mountedis fixed detachably by screws to the upper end of the rotation shaft2119.

The objective lens 2125 is located on a straight line extending from therotation shaft 2119 in a moving direction of the horizontally movingmember 2116. The objective lens 2125 is supported by the verticallymoving member 2126. The vertically moving member 2126 is supported sothat it can be moved vertically by the straight moving guide 2124 fixedto the upper base member 2114. Further, the vertically moving member2126 is coupled to, through the ball screw 2127, the stepping motor 2128fixed to the upper base member 2114, and moved vertically by thestepping motor 2128. The straight moving guide 2124, the verticallymoving member 2126, the ball screw 2127 and the stepping motor 2128 areall provided under the upper base member 2114.

The moving device thus configured enables the relative movement of thespecimen 2123 mounted on the table 2122 and the objective lens 2125.That is, the horizontally moving member 2116 can move linearly in onedirection in the horizontal plane relative to the upper base member2114. Further, the table 2122 can make rocking movement with respect tothe horizontally moving member 2116. Moreover, the vertically movingmember 2126 can move linearly in a vertical direction with respect tothe upper base member 2114. That is, the specimen 2123 can move linearlyin one direction and rock with respect to the objective lens 2125, andthe objective lens 2125 can move relatively in a vertical direction withrespect to the specimen 2123. As a result, the specimens 2123 can beobserved. Moreover, because the image pickup device 2150 is locatedoutside the culture device 2101, noise resulting from the temperature ofthe image pickup device 2150 can be reduced. Further, the upper basemember 2114 and the lower base member 2191 that have differenttemperatures inside and outside the culture device 2101 are made of thelow expansion material, such that distortion due to thermal expansioncan be reduced and the adjustment of the optical system is notdisturbed.

[Outline of Culture Device]

The culture device 2101 comprises the upper base member 2114, a door2101 a located above the upper base member 2114, and a base portion 2101b located under the upper base member 2114. The door 2101 a can open andclose with respect to the upper base member 2114 to mount the specimen2123 on the table 2122. When the door 2101 a is closed, the upper basemember 2114 and the door 2101 a define a culture space. In order to keepan airtight state between the door 2101 a and the upper base member 2114when the door 2101 a is closed, an elastic seal member 2112 is providedbetween the door 2101 a and the upper base member 2114. The base portion2101 b is held by the support columns 2111, and the elastic seal member2112 is provided between the base portion 2101 b and the upper basemember 2114 to keep an airtight state between the base portion 2101 band the upper base member 2114.

The culture device 2101 comprises an insulating material 2102, ametallic interior portion 2103 made of stainless steel havingantibacterial and corrosion resistance properties or anti-bacteriallycoated, a sensor 2104 to sense the temperature, humidity and PH in theculture device, a heater 2105 provided in the interior of the culturedevice for internal temperature adjustment, humidification means or ahumidification heater 2106 provided on the upper base member 2114 fortemperature adjustment in the culture device, an electromagnetic valve2107 that is connected to a carbon dioxide gas cylinder to adjust thecarbon dioxide gas concentration for PH adjustment and that suppliescarbon dioxide gas, a column heater 2108 to adjust the temperature ofthe support columns 2111 coupling the inside and outside of the culturedevice 2101, and a support column sensor 2109 to measure the temperatureof the support columns 2111. On the upper base member 2114, ahumidification pad 2113 containing water for humidification in theculture device 2101 is placed at a position above the humidificationheater 2106. The culture device 2101 further comprises a controller 2110to perform an operation for maintenance of a set condition in accordancewith a signal from the sensor 2104 in order to control the heater 2105,the humidification heater 2106 and the electromagnetic valve 2107.

According to such a configuration, the insulating material 2102thermally isolates the image pickup device 2150 from the culture device2101. The insulating material 2102 and the seal member 2112 reduce heatgoing in and out of the culture device 2101. This reduces the influenceof outdoor air temperature changes and enables the stabilization oftemperature. Moreover, as the less heat goes in and out, the capacity ofthe heater 2105 and the humidification heater 2106 and water capacity ofthe humidification pad 2113 can be reduced. Further, as plenty ofnutrients are provided to the culture medium where the cells arecultured, microorganisms having high reproduction ability infiltratingtherein cause an adverse effect to the cultured cells. However, theinterior portion 2103 of the culture device 2101 having theantibacterial and corrosion resistance properties prevents theinfiltration of the microorganisms and can maintain the activity of thecultured cells.

Furthermore, high humidity is maintained in the culture space of theculture device 2101 to prevent the culture medium from being dried dueto the evaporation of water. Therefore, if the specimen operated at aroom temperature of about 23° C. is brought into the culture device,condensation is caused on the outside of the container, resulting in thedeterioration of the observation image through the microscope. It isthus preferable that humidification control described below is performedfor the culture device 2101. The culture device 2101 has a door sensor,which is not specifically shown, to sense that the door 2101 a is openedor closed. In accordance with a signal (an instruction to mount thespecimen 2123) from the door sensor, the controller 2110 causes thehumidification heater 2106 to warm the humidification pad 2113 to starthumidification after a certain period of time has passed (e.g., afterten minutes) so that the cultured cells including the container (i.e.,the specimen 2123) will be at the same temperature as that inside theculture device 2101. Owing to this control, the cultured cells arehumidified after reaching the same temperature as that inside theculture device 2101, whereby a satisfactory microscope observation imagecan be obtained without causing condensation in the cultured cellsincluding the container. Moreover, if a specimen mounting button isprovided in the controller 2110 instead of the door sensor and thecontroller 2110 performs similar humidification control in accordancewith an instruction based on the specimen mounting button, similareffects can be provided. In addition, instead of turning thehumidification heater 2106 on and off, a door provided for thehumidification pad 2113 may be opened and closed.

[Details of Table Section and Objective Lens Section of MicroscopeDevice]

FIG. 9 shows in an enlarged manner a peripheral part of the table shownin FIG. 8. Between the table 2122 and the upper base member 2114, anintermediate member 2129 is located fixedly to the horizontally movingmember 2116. Between the table 2122 and the intermediate member 2129,there are provided two ring-shaped seat seals 2199 made of ethylenetetrafluoride (PFTE), and an elastic O-ring 2130. The O-ring 2130 islocated in a pressed state. Also, between the upper base member 2114 andthe intermediate member 2129, there are provided the two seat seals 2199and the O-ring 2130, and the O-ring 2130 is located in a pressed state.

FIG. 10 shows in an enlarged manner a peripheral part of the objectivelens shown in FIG. 8. The upper base member 2114 has an opening 2114 bto protrude the objective lens 2125, and a groove is formed on an innerperipheral surface of the opening 2114 b, and an elastic O-ring 2134 isreceived in the groove of the opening 2114 b. In such a state that theobjective lens 2125 protrudes out of an upper surface of the upper basemember 2114 through the opening 2114 b, the O-ring 2134 is pressed.

In FIG. 9, the rotation of the stepping motor 2121 rocks the table 2122.The table 2122 rocks such that sliding is mainly caused between the twoseat seals 2199 of the PFTE material having a low friction coefficient.Even if a distance change is caused between the table 2122 and theintermediate member 2129 through the seat seals 2199, the O-ring 2130elastically deforms to absorb the distance change to always prevent gapformation. Similarly, even if a distance change is caused between theupper base member 2114 and the intermediate member 2129 through the seatseals 2199, the O-ring 2130 works similarly to prevent the gapformation. Moreover, as the seat seals 2199 have a low frictioncoefficient, sliding resistance is kept low. Even if membersconstituting the guides and stages have lower rigidity, accuratemovement is possible. Further, low frictional force can prevent theabrasion of the members.

Furthermore, in FIG. 10, a distance change is also caused between theobjective lens 2125 and the opening 2114 b of the upper base member 2114in the vertical movement of the objective lens 2125, but the O-ring 2134elastically deforms to absorb the distance change to always prevent gapformation.

In FIG. 8, if the door 2101 a is closed, the seal member 2112 is pressedbetween the door 2101 a and the upper base member 2114. Therefore, nogap is formed between the door 2101 a, that is, the culture device 2101and the upper base member 2114.

Thus, the microscope device is isolated from the moisture of the culturedevice 2101 by the upper base member 2114, the base portion 2101 b andsealing structures (the seal member 2112, the seat seals 2199, theO-ring 2130 and the O-ring 2134). In other words, the upper base member2114, the base portion 2101 b and the sealing structures (the sealmember 2112, the seat seals 2199, the O-ring 2130 and the O-ring 2134)constitute isolation means or an isolator to isolate the microscopedevice from the moisture of the culture device 2101.

This configuration can keep the culture space defined by the closed door2101 a and the upper base member 2114 from the outside air. Further, thetable 2122 and the objective lens 2125 inside the culture space can bemoved from the outside of the culture space. It is thus easy to maintainthe temperature and humidity of the culture space, so that necessaryheater capacity and a necessary amount of water can be reduced. It isfurther possible to reduce the infiltration of moisture into a mechanismpart (the moving device) and an optical part, enabling the prevention ofrust and condensation.

Furthermore, the use of PTFE having a low friction coefficient for asealing surface enables a microscope having both positional accuracy andsealing performance.

Even with the above-described sealing structures (the seal member 2112,the seat seals 2199, the O-ring 2130 and the O-ring 2134), moistureslightly infiltrates into a lower surface side of the upper base member2114. In order not to expose the mechanism part (the moving device) andthe optical part to such moisture, in FIG. 9, the gap between the lowersurface of the upper base member 2114 and the horizontally moving member2116 is set at 0.1 mm or less, and an intake pipe 2131 and an exhaustpipe 2132 are connected to the horizontally moving member 2116. Theintake pipe 2131 and the exhaust pipe 2132 are led to the external, andan air pressure source is connected to the intake pipe 2131 for airintake. The intake pipe 2131 is wound several times in the culturedevice 2101 to elongate a passage in the culture device 2101. The intakepipe 2131 and the exhaust pipe 2132 constitute dehumidifying means or adehumidifier that connects the atmosphere in the microscope device tothe air outside the culture microscope apparatus and that dehumidifiesthe atmosphere in the microscope device. Further, the intermediatemember 2129 comprises, at a cylindrical portion connected to thehorizontally moving member 2116, a communication hole 2129 a penetratingthe cylindrical portion.

In FIG. 10, the upper base member 2114 is provided with a capturingmember 2133 extending from the upper base member 2114 in the axialdirection of the objective lens 2125. The capturing member 2133 has anopening through which the objective lens 2125 passes, and the O-ring2134 is mounted in the groove formed in the inner peripheral surface ofthe opening to press the objective lens 2125. The above-mentioned intakepipe 2131 and the exhaust pipe 2132 are also connected between theO-ring 2134 of the capturing member 2133 and the O-ring 2134 of theupper base member 2114.

The moisture that has infiltrated from the culture space defined by theclosed door 2101 a and the upper base member 2114 through the spacebetween the two seat seals 2199 diffuses between the upper base member2114 and the horizontally moving member 2116 from a gap between theintermediate member 2129 and the rotation shaft 2119 through thecommunication hole 2129 a. The moisture diffused between the upper basemember 2114 and the horizontally moving member 2116 is discharged to theoutside from the exhaust pipe 2132 together with the outside airintroduced from the intake pipe 2131. The outside air introduced fromthe intake pipe is warmed by the long passage in the culture device 2101and reaches the horizontally moving member 2116 without dropping thetemperature of the members. The moisture that has infiltrated from thespace between the objective lens 2125 and the O-ring 2134 is similarlydischarged to the outside.

The configuration described above also forces the slight amount ofmoisture that has infiltrated from each of the sealing structures (theseat seals 2199, the O-ring 2130 and the O-ring 2134) to be dischargedto the outside. Thus, moisture does not reach the mechanism part (themoving device) and the optical part without the fear of rust andcondensation. Further, a constant temperature of the objective lens 2125can eliminate focal movement of the objective lens 2125 due totemperature changes. This enables a long-term observation withoutdefocusing.

[Manipulator]

Next, a manipulator to introduce genes and drugs into the cultured cellswill be described referring to FIG. 10. The manipulator has an arm 2142capable of rocking and vertical movement, and the arm 2142 holds asyringe 2143 at an end. The arm 2142 is detachably fixed to a verticallymoving shaft 2138 by screws. The vertically moving shaft 2138 isreceived in a cylindrical rotation shaft 2136 and can move verticallywith respect to the rotation shaft 2136. The rotation shaft 2136 isattached to the upper base member 2114 through a bearing 2137 and canrotate with respect to the upper base member 2114. The rotation shaft2136 comprises a main wheel 2195 at a lower end. The main wheel 2195engages with a pinion 2193 attached to an output shaft of a steppingmotor 2194 fixed to the upper base member 2114. The vertically movingshaft 2138 has a female screw at a lower end. The female screw of thevertically moving shaft 2138 engages with a male screw formed in theoutput shaft of a stepping motor 2140. The vertically moving shaft 2138has a groove 2138 a extending vertically in an outer peripheral surface.A pin 2139 fixed to the rotation shaft 2136 is received in the groove2138 a. The pin 2139 determines the range of vertical movement of thevertically moving shaft 2138 with respect to the rotation shaft 2136,and regulates the rotation of the vertically moving shaft 2138 withrespect to the rotation shaft 2136. A position to fix the arm 2142 tothe vertically moving shaft 2138 is adjusted so that the distancebetween the syringe 2143 and a core of the rotation shaft 2136 will bethe same as the distance between an optical axis of the objective lens2125 and the core of the rotation shaft 2136.

In the configuration described above, the arm 2142 can be moved tolocate the syringe 2143 at the cells in the center of a viewing fieldeven in a highly humid environment, and for example, a reagent placed onthe upper base member 2114 can be administered to the cultured cells.Further, the stepping motor 2194 to rotate the arm 2142 and the steppingmotor 2118 to horizontally move the specimen 2123 can be driven inconjunction with each other to locate the syringe 2143 at an optionalposition in a predetermined range.

In the culture microscope apparatus of the present embodiment, when thetable 2122 and the arm 2142 are removed, the only members protruding onthe upper side of the upper base member 2114 are cylindrical members(the rotation shaft 2119 and the intermediate member 2129 of the movingdevice, the rotation shaft 2136 of the manipulator), so that cleaningcan be easily performed.

[Water Supply]

A water supply device for a water-immersion objective lens will bedescribed referring to FIG. 10. The culture microscope apparatus of thepresent embodiment comprises the water supply device, which supplieswater to the water-immersion objective lens, considering the case wherethe objective lens 2125 is a water-immersion objective lens. The watersupply device comprises a cooler capable of setting a temperaturedifferent from a set temperature of the culture device 2101. The coolercomprises, but not limited to, a peltier element 2144 in the presentembodiment. On a lower surface of the table 2122, the peltier element2144 is fixed at a place where it can be located on the optical axis ofthe objective lens 2125 when the table 2122 moves. The peltier element2144 has a water supply surface 2144 a that can face the objective lens2125. The culture space is maintained in a saturated state close to arelative humidity of 100%, so that if the water supply surface 2144 a ofthe peltier element 2144 is cooled off several times, steam is condensedon the water supply surface 2144 a. The table 2122 is moved by theabove-mentioned moving device to locate the water supply surface 2144 aabove the objective lens 2125 and then the table 2122 is lowered suchthat water condensed on the water supply surface 2144 a can be suppliedto the objective lens 2125. According to this configuration, water canbe supplied to the objective lens 2125 only by the peltier element 2144without using an extra member. Thus, an inexpensive water supply devicecan be provided.

The peltier element 2144 can be provided not on the table 2122 but onthe upper surface of the upper base member 2114, and the above-mentionedmanipulator can be used to supply water condensed on the upper surfaceof the peltier element 2144 to the objective lens 2125. Thisconfiguration can reduce the temperature changes of the specimen becausethe peltier element 2144 is not provided on the table 2122.

[Dark Field]

The microscope device is capable of fluorescent observation and darkfield observation. The fluorescent observation is used to identify theexpression of fluorescent protein at a target part, and the dark fieldobservation is used to visualize the nucleus and outline of cells forconfirmation of the positions of cells, the state of culture orbacterial contamination.

The microscope device includes an illumination device for illuminatingthe cultured cells and an observation device to observe the culturedcells. FIG. 11 shows the illumination device applicable to themicroscope device shown in FIG. 8. As shown in FIG. 11, the illuminationdevice comprises light emitting diodes 2145 having different emissionwavelengths, excitation filters 2146 located in front of the lightemitting diodes 2145, and a bending member 2147. The light emittingdiodes 2145, the excitation filters 2146 and the bending member 2147 areall located at an outer peripheral part of the objective lens 2125. Theexcitation filters 2146 selectively transmit light having a specificwavelength among wavelengths of illumination light emitted from thelight emitting diodes 2145. The bending member 2147 bends theillumination light that has penetrated the excitation filters 2146 andorients it toward the specimen 2123.

As shown in FIG. 8, the observation device comprises the objective lens2125, an image-forming lens 2149, which cooperates with the objectivelens 2125 to constitute an image-forming optical system, the imagepickup device 2150, which picks up an optical image formed by theimage-forming optical system, and a monitor 2192 to display the imageobtained by the image pickup device 2150. The base portion 2101 b isprovided with an optical window 2223 so that light from the specimen2123 travels to the image pickup device 2150 through the objective lens2125 and the image-forming lens 2149. The optical window 2223 maycomprise a transparent optical member such as glass plate. Theobservation device further comprises an emission filter 2148, whichselectively transmits light having a specific wavelength amongwavelengths of observation light directed to the image pickup device2150, and a turret 2151 to locate the emission filter 2148 on theoptical axis as required. The image pickup device 2150 is preferably acooled CCD considering the fluorescent observation.

In the dark field observation, in FIG. 8, the turret 2151 is switched tolocate an air hole on the optical axis and displace the emission filter2148 from the optical axis. In FIG. 11, the light emitted by the lightemitting diodes 2145 penetrates the excitation filters 2146, andilluminates the specimen 2123 due to the bending member 2147 from theoutside of NA of the objective lens 2125. Therefore, the illuminationlight and the light regularly reflected by the lower surface of thespecimen container are not captured by the objective lens 2125. Thereflected light and scattered light alone due to the cultured cells inthe specimen 2123 are captured by the objective lens 2125 and detectedby the image pickup device 2150. Thus, even transparent cultured cellscan be visualized without being dyed. Further, because a generally usedtransmitted-light illumination portion used in phase differenceobservation is not necessary, a space is produced above the specimen,thus facilitating operations including taking the specimen 2123 in andout of the culture device 2101 and administering the reagent to thespecimen 2123.

FIG. 12 shows another illumination device applicable to the microscopedevice shown in FIG. 8. If NA of the objective lens 2125 is, forexample, 0.85, the range of light captured by the objective lens 2125will be about 60 degrees from the optical axis. Illumination opticalaxes are set on a bus at 70 degrees from the optical axis toward thecenter of a specimen container bottom surface, and an excitation filter2152, a collimating lens 2153 and a light emitting diode 2154 arelocated on the illumination optical axes. The excitation filter 2152,the collimating lens 2153 and the light emitting diode 2154 are sealedby a dustproof glass 2156 inside an illumination member 2155 providedout of a moving range of the table 2122 on the upper base member 2114.

In other words, the illumination device of FIG. 12 comprises the lightemitting diode 2154 to emit the illumination light, the collimating lens2153 to form the illumination light emitted by the light emitting diode2154 into parallel light, the excitation filter 2152, which selectivelytransmits light having a specific wavelength among wavelengths of theillumination light emitted from the light emitting diode 2154, theillumination member 2155 housing the light emitting diode 2154, thecollimating lens 2153 and the excitation filter 2152, and the dustproofglass 2156 constituting an optical window provided in the illuminationmember 2155. The illumination optical axis passing the light emittingdiode 2154, the collimating lens 2153 and the excitation filter 2152 isinclined at 70 degrees with respect to the optical axis of the objectivelens 2125.

The light emitted by the light emitting diode 2154 is brought intoparallel light by the collimating lens 2153, and illuminates uniformlywithin an observation field in the objective lens 2125. The light thathas penetrated the specimen 2123 is the light outside the NA of theobjective lens 2125, and is therefore not captured by the objective lens2125. The reflected light and scattered light alone due to the specimen2123 are captured by the objective lens 2125 and detected by the imagepickup device 2150. Therefore, effects similar to those in dark fieldillumination by the illumination device shown in FIG. 11 are provided;for example, even transparent cultured cells can be visualized withoutbeing dyed, and the transmitted-light illumination portion is notnecessary. When the fluorescent observation is not performed together orwhen narrow-band wavelengths are not necessary in an excitationwavelength, the excitation filter 2152 may be removed from theconfiguration.

[Fluorescence]

The fluorescent observation can also be performed using the illuminationdevice in FIG. 11. In the fluorescent observation, in FIG. 8, the turret2151 is switched to locate on the optical axis the emission filter 2148adapted to a fluorescent wavelength of the specimen 2123. In FIG. 11,light having a wavelength that is needed to excite the specimen amongwavelengths of light emitted from the light emitting diode 2154 isselectively transmitted by the excitation filter 2146 and illuminatesthe specimen 2123. The specimen 2123 excited by the illumination lightemits fluorescence light having a wavelength longer than the wavelengthused for the excitation. The fluorescence light is captured by theobjective lens 2125, brought into parallel light and exits from theobjective lens 2125 to reach the emission filter 2148. The light thathas penetrated the emission filter 2148 is imaged on a light receivingsurface of the image pickup device 2150 by the image-forming lens 2149,and displays an object image on the monitor 2192. Owing to the darkfield observation, the illumination light is not captured by theobjective lens 2125, so that SN in accordance with auto-fluorescence inthe objective lens 2125 is not reduced by the illumination light.

The fluorescent observation can also be performed using the illuminationdevice in FIG. 12. In FIG. 12, the light emitted by the light emittingdiode 2154 is brought into parallel light by the collimating lens 2153,and the light needed to excite the specimen is selectively transmittedby the excitation filter 2152 and illuminates the specimen uniformly.The function leading to the image-forming is the same as in theillumination device in FIG. 11.

Furthermore, instead of the light emitting diode 2154, a light sourceprovided outside the apparatus may be used in such a manner to transmitthrough a fiber. When the fiber is used, a high luminance light sourcecan be used because it is not necessary to consider the intensity oflight and heat of the light source.

Here, the illumination device for the fluorescent observation is anoblique illumination device as shown in FIG. 11 and FIG. 12, but it mayalso be an incident-light illumination device. That is, the illuminationdevice for the fluorescent observation includes the objective lens 2125and may have the configuration similar to those of the illuminationdevices described in the first embodiment to fourth embodiment.

[Phase Difference]

FIG. 13 shows a modification of the culture microscope apparatus of thepresent embodiment. More specifically, the culture microscope apparatusthat has changed the microscope device shown in FIG. 8 from theconfiguration suitable for the dark field observation to theconfiguration suitable for the phase difference observation is shown. InFIG. 13, members indicated by the same numerals as the members shown inFIG. 8 are the same and will not be described in detail.

In the culture microscope apparatus of the present modification, themicroscope device comprises a phase difference objective lens 2157 inplace of the objective lens 2125 in FIG. 8 and also comprises atransmitted-light illumination device in place of the dark fieldillumination device in FIG. 11 and FIG. 12, as understood from FIG. 13.

The transmitted-light illumination device comprises an illuminationsupport column 2158, a ring slit 2159, a light emitting diode 2160, areflecting mirror 2161 and a collimating lens 2162. The illuminationsupport column 2158 is placed out of the moving range of the table 2122on the upper base member 2114. The illumination support column 2158houses the ring slit 2159, the light emitting diode 2160 and thereflecting mirror 2161. The collimating lens 2162 is inserted into anopening of the illumination support column 2158 and closely fixed by anadhesive material. The ring slit 2159 has a ring-shaped opening and islocated at a position conjugate with a rear focal plane of the phasedifference objective lens 2157. The light emitting diode 2160 is locatedin the vicinity of the ring slit 2159.

The phase difference objective lens 2157 comprises a phase plate 2163 onthe rear focal plane. The size of the phase plate 2163 includes theprojected ring slit 2159. That is, an image of the ring slit 2159 isprojected on an inner side of the phase plate 2163. Moreover, the phaseplate 2163 comprises an optical member that shifts the phase oftransmitted light by ¼ wavelengths, and an ND film that attenuates thetransmitted light.

Light emitted from the light emitting diode 2160 passes through theopening of the ring slit 2159, has its direction changed by thereflecting mirror 2161, is brought into parallel light by thecollimating lens 2162, and illuminates the specimen 2123 uniformly.

Zero-th light that has penetrated the specimen 2123 converges on thephase plate 2163 of the phase difference objective lens 2157, and issubjected to phase shift and light amount attenuation. Further, primarylight diffracted at the specimen 2123 does not converge on the phaseplate 2163 on the rear focal plane of the phase difference objectivelens 2157, and is not therefore subjected to phase shift and lightamount attenuation.

The zero-th light and the primary light are imaged on a light receivingsurface of the image pickup device 2150 by the image-forming lens 2149.The phase shift of the zero-th light performed by the phase plate 2163causes interference between the zero-th light and the primary light, sothat even undyed specimens can be observed. Moreover, the collimatinglens 2162 shuts off the culture space from the inside of theillumination support column 2158, so that condensation is not caused onoptical members inside the illumination support column.

<Another Modification of Fifth Embodiment>

FIG. 15 shows another modification of the culture microscope apparatusof the fifth embodiment of the present invention. More specifically, theculture microscope apparatus is shown in which a dimmer unit is added tothe culture microscope apparatus shown in FIG. 8. In FIG. 15, membersindicated by the same numerals as the members shown in FIG. 8 are thesame and will not be described in detail.

In the culture microscope apparatus of the present modification, adimmer unit 2188 is located above the objective lens 2125. The dimmerunit 2188 faces the objective lens 2125, and the specimen 2123 to beobserved is properly located between the dimmer unit 2188 and theobjective lens 2125. The dimmer unit 2188 is supported by a supportcolumn 2189, and the support column 2189 is fixed to the upper basemember 2114 out of the moving range of the table 2122. The dimmer unit2188 may comprise the dimmer unit described in the first embodiment tofourth embodiment. That is, the dimmer unit 2188 has the sameconfiguration as those of the dimmer unit 1122, the dimmer unit 1201 orthe dimmer unit 1301.

According to the present modification, it is possible to significantlyreduce the discoloration of the fluorescent dyes that are not targetedfor observation, and to observe the fluorescent image with good contrastfor a long period of time.

<Still Another Modification of Fifth Embodiment>

FIG. 15 shows still another modification of the culture microscopeapparatus of the fifth embodiment of the present invention. In FIG. 15,members indicated by the same numerals as the members shown in FIG. 8are the same and will not be described in detail.

The upper base member 2114 in the culture device 2101 is set up with aleg attachment portion 2202 fixed through support columns 2201, and thecenter of the leg attachment portion 2202 and the lower base member 2191are connected by a leg portion 2205.

Furthermore, the following member is used as a seal member 2203 of theobjective lens 2125. The seal member 2203 is a thin elastic rubbermaterial, and has a cylindrical portion and two planar portions. The twoplanar portions are fixed to the objective lens 2125 and the upper basemember 2114 through a fixing member 2204.

The leg attachment portion 2202 and the lower base member 2191 that havedifferent temperature settings inside and outside the culture device2101 are connected by the leg portion 2205, so that the distortion ofthe material caused by an expansion difference due to the temperaturecan be reduced and the optical adjustment is not disturbed.

Furthermore, the seal member 2203 enables sealing with a low slidingresistance, allowing an improvement in positional repeatability.

<Further Still Another Modification of Fifth Embodiment>

FIG. 16 shows further still another modification of the culturemicroscope apparatus of the fifth embodiment of the present invention.In FIG. 16, members indicated by the same numerals as the members shownin FIG. 8 are the same and will not be described in detail.

The culture microscope apparatus of the present modification furthercomprises a transmitted-light illumination device 2210 fortransmitted-light illumination of the specimen 2123, in addition to theconstruction of the culture microscope apparatus shown in FIG. 8. Thetransmitted-light illumination device 2210, which is located above theobjective lens 2125, is attached to the door 2101 a so as to be detachedfrom the door 2101 a. The door 2101 a is provided with two opticalwindows 2221 and 2222, which transmit light from the transmitted-lightillumination device 2210. The optical windows 2221 and 2222 may comprisetransparent optical members such as glass plates.

The culture microscope apparatus has a region I that is basicallydefined by the door 2101 a and the upper base member 2114, a region IIthat is basically defined by the base portion 2101 b and the upper basemember 2114, and a region III under the culture device 2101. In theregion I, the temperature and humidity are maintained at a level equalto that in an environment inside a body of a human being, an animal or aplant. In the region II, the temperature is on a level equal that of theregion I and the humidity is maintained at a normal humidity level. Inthe region III, the temperature and humidity are on a normal temperatureand normal humidity level.

The regions I, II and III are located along the optical axis of theimage-forming optical system including the objective lens 2125, theimage-forming lens 2149 and the image pickup device 2150. The specimen2123 is located in the region I, and the image pickup device 2150 in theregion III.

The regions II and III are optically connected through the opticalwindow 2223, which is positioned between the regions II and III, so thatlight for the image-forming optical system, light from the specimen2123, passes through a boundary between the regions II and III. Theregion I is optically connected to the transmitted-light illuminationdevice 2210 through the optical windows 2221 and 2222, which arepositioned between the region I and the transmitted-light illuminationdevice 2210, so that light for the image-forming optical system, lightfrom the transmitted-light illumination device 2210, passes into theregion I.

The regions I and II satisfy the following conditional equations:36° C.≦t1≦38° C., 90%≦h1≦100%36° C.≦t2≦38° C., 50%≦h2≦80%where t1 indicates the temperature of the region I, h1 indicates thehumidity of the region I, t2 indicates the temperature of the region II,and h2 indicates the humidity of the region II.

The specimen 2123 and the objective lens 2125, which are weak in heatretaining and moisture retaining performance, are preferably separatedfrom the normal temperature and normal humidity environment. An adverseeffect (especially condensation) on the objective lens 2125 due to theculture environment is desirably reduced as much as possible. The imagepickup device 2150 in the image-forming optical system, which isbasically heating elements, is desirably located as far away from theculture environment as possible.

The culture microscope apparatus of the present modification has theregion II as a buffer region, through which the specimen 2123 and theimage pickup device 2150 are positioned and in which the objective lens2125 is mainly located, so that the adverse effect (especiallycondensation) on the objective lens 2125 is effectively reduced and theculture environment is easily maintained in preferable condition.

Sixth Embodiment

The present embodiment is also directed to the culture microscopeapparatus that causes a small change in the installed environment andthat is easily cleaned, similarly to the fifth embodiment.

FIG. 17 schematically shows the culture microscope apparatus of a sixthembodiment of the present invention.

The culture microscope apparatus of the present embodiment comprises anincubator 2164 as a culture device, which can control temperature,humidity and PH and is used for the cultured cells, a microscope device2165 housed in the incubator 2164, and a slide device 2166 that movesthe microscope device 2165 between the inside and outside of theincubator 2164. The slide device 2166 is provided in the incubator 2164.

The microscope device 2165 comprises an illumination device 2167 forilluminating the cultured cells, an observation device 2168 to observethe cultured cells, a moving device 2169, and a microscope container2170. The illumination device 2167 comprises a light emitting diode2171, a collimating lens 2172, and an excitation filter 2173. Theobservation device 2168 comprises an objective lens 2174, a turret 2176equipped with an emission filter 2175, an image-forming lens 2177, andan image pickup device 2178. The objective lens 2174 and the imagepickup device 2178 constitute an image-forming optical system. Themoving device relatively moves the specimen and the objective lens 2174placed in the microscope container 2170, and comprises a horizontalstage 2179 capable of two-dimensional movement in a horizontal plane anda vertical stage 2180 placed on the horizontal stage 2179 and capable ofvertical movement.

The objective lens 2174 and the illumination device 2167 are integrallyfixed to the vertical stage 2180. The emission filter 2175, the turret2176, the image-forming lens 2177 and the image pickup device 2178 areintegrally fixed to the horizontal stage. The microscope container 2170is substantially a rectangular-parallelepiped-shaped box having twoopenings. One opening is located on an upper surface of the microscopecontainer 2170, while the other opening is located on a side surface ofthe microscope container 2170. An elastic O-ring 2181 is placed in agroove provided in the vicinity of the opening on the upper surface, andan optically transparent glass plate 2182 is fixed to the microscopecontainer 2170 in a state pressing the O-ring 2181. The glass plate 2182and the O-ring 2181 cooperate with the microscope container 2170 toconstitute isolation means or an isolator to isolate the microscopedevice from the moisture of the culture device.

A communication pipe 2183 is fixed to the opening on the side surface ofthe microscope container 2170. The communication pipe 2183 is anextensible bellows, and connects the inside of the microscope container2170 to the outside of the incubator 2164. The communication pipe 2183is schematically drawn so that it extends in a direction vertical to themoving direction, that is, longitudinal direction of the microscopecontainer 2170, that is, in the lateral direction for convenience ofdrawing in FIG. 17, but it is actually provided so that it extends inparallel with the moving direction, that is, longitudinal direction ofthe microscope container 2170. In addition, one communication pipe 2183is drawn in FIG. 17, but two communication pipes 2183 are actuallyprovided for air intake and exhaustion. The slide device 2166 comprisesa fixed portion 2184, a moving portion 2185 and a rolling portion 2186,and the fixed portion 2184 is fixed to the incubator 2164, and themoving portion 2185 is fixed to the microscope container 2170.

The incubator 2164 is provided with a fan 2231 for the purpose ofeliminating uneven temperature and humidity inside of the incubator2164. However, vibration due to the fan 2231 deteriorates an obtainedimage. Therefore, the controller 2232 performs blurring preventioncontrol to stop the fan when an image is taken. The blurring preventioncontrol may be started simultaneously with the lighting of theillumination device 2167. This blurring prevention control makes itpossible to obtain high-quality image without blurring the image due tothe fan vibration.

Owing to the sealing structure using the O-ring 2181, the infiltrationof moisture into the microscope container 2170 is reduced in anenvironment with the highly humid incubator 2164, and even a slightamount of infiltrated moisture is discharged outside by thecommunication pipes 2183 without causing rust and condensation in themicroscope part. Moreover, the slide device 2166 is provided to make iteasy to take the microscope container 2170 in and out of the incubator2164 for easier cleaning, thereby enabling contamination prevention evenin the cell culture where contamination of bacteria and molds is notpreferred. Further, a slight amount of moisture infiltration is causedin a sealed portion with the O-ring, so that two communication pipes maybe provided to introduce the outside air through one communication pipeand to compulsorily remove the moisture inside through the othercommunication pipe. This enables a more complete moisture measurement.

The culture microscope apparatus has a region I between the incubator2164 and the microscope container 2170, a region II within themicroscope container 2170, and a region III out of the incubator 2164.In the region I, the temperature and humidity are maintained at a levelequal to that in an environment inside a body of a human being, ananimal or a plant. In the region II, the temperature is on a level equalthat of the region I and the humidity is maintained at a normal humiditylevel. In the region III, the temperature and humidity are on a normaltemperature and normal humidity level.

The regions I, II and III are located along the optical axis of theimage-forming optical system of the observation device 2168. Thespecimen 2123 is located in the region I.

The regions I and II are optically connected through the opticallytransparent glass plate 2182, which is positioned between the regions Iand II, so that light for the observation device 2168, light from thespecimen 2123, passes through a boundary between the regions I and II.

The regions I and II, also in the present embodiment, satisfy thefollowing conditional equations:36° C.≦t1≦38° C., 90%≦h1≦100%36° C.≦t2≦38° C., 50%≦h2≦80%where t1 indicates the temperature of the region I, h1 indicates thehumidity of the region I, t2 indicates the temperature of the region II,and h2 indicates the humidity of the region II.

The culture microscope apparatus of the present embodiment has theregion II, in which the observation device 2168 is located, so that theadverse effect (especially condensation) on the observation device 2168is effectively reduced.

In the present embodiment, the microscope container 2170 is taken in andout of the incubator 2164 by the slide device 2166, but the mechanism totake the microscope container 2170 in and out is not limited thereto,and any known moving mechanism is applicable. Moreover, the movingmechanism such as the slide device 2166 may be omitted, and themicroscope container 2170 may be manually taken in and out of theincubator 2164.

In the present embodiment, the illumination device 2167 for thefluorescent observation is again the oblique illumination device, butmay be the incident-light illumination device. That is, the illuminationdevice 2167 for the fluorescent observation includes the objective lens2174 and may have the configuration similar to those of the illuminationdevices described in the first embodiment to fourth embodiment.

While the embodiments of the present invention have been described abovereferring to the drawings, the present invention is not limited theseembodiments, and various modifications and alterations may be madewithout departing from the sprit thereof.

The microscope device in the culture microscope apparatus is an invertedmicroscope in the above embodiments, but is not limited to it, that is,the microscope device may be an up-right microscope.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details and representative embodiments shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventionconcept as defined by the appended claims and their equivalents.

1. A culture microscope apparatus comprising: (i) a microscope devicefor observing a specimen, the microscope device comprising: an objectivelens for allowing the specimen to be observed, a moving device forrelatively moving the specimen and the objective lens, an image pickupdevice for picking up an image of the specimen through the objectivelens, an upper base member on which the objective lens and the movingdevice are placed, and a lower base member on which the image pickupdevice is placed, the upper base member and the lower base member beingconnected by support columns; (ii) a culture device capable ofcontrolling temperature and humidity, the culture device comprising anisolation wall including an insulating material that thermally isolatesthe image pickup device from the culture device; and (iii) isolationmeans for isolating the microscope device from moisture of the culturedevice.
 2. The culture microscope apparatus according to claim 1,wherein the microscope device further comprises: an illumination devicefor illuminating the specimen, an excitation filter for selectivelytransmitting light having only a specific wavelength among wavelengthsof illumination light emitted from the illumination device, an emissionfilter for selectively transmitting light having only a specificwavelength among wavelengths of observation light directed to the imagepickup device, a ring slit located in the illumination device at aposition conjugate with a rear focal plane of the objective lens, and aring-shaped phase plate located on the rear focal plane of the objectivelens.
 3. The culture microscope apparatus according to claim 1, whereinthe microscope device further comprises: an illumination device forilluminating the specimen, an excitation filter for selectivelytransmitting light having only a specific wavelength among wavelengthsof illumination light emitted from the illumination device, and anemission filter for selectively transmitting light having only aspecific wavelength among wavelengths of observation light directed tothe image pickup device, and the illumination device illuminates thespecimen from outside of an aperture of the objective lens.
 4. Theculture microscope apparatus according to claim 1, wherein the isolationmeans comprises an optically transparent glass.
 5. The culturemicroscope apparatus according to claim 1, wherein the microscope devicecomprises dehumidifying means, connecting an atmosphere in themicroscope device to an air outside the culture microscope apparatus,for dehumidifying the atmosphere in the microscope device.
 6. Theculture microscope apparatus according to claim 1, wherein the movingdevice includes a table on which the specimen is mounted and a driveportion to move the table, and wherein the isolation means includes aplate-like isolation wall having a through-hole through which the driveportion partially passes, and a sealing structure provided between themoving device and the isolation wall.
 7. The culture microscopeapparatus according to claim 6, wherein the sealing structure includes aseat seal made of ethylene tetrafluoride and an elastic member to pressthe seat seal.
 8. The culture microscope apparatus according to claim 1,comprising a fan for air circulation in the culture device and acontroller to control the fan, wherein the controller stops the fan ofthe culture device when an image is taken.
 9. The culture microscopeapparatus according to claim 1, comprising a humidification means forhumidity adjustment in the culture device, and a controller forcontrolling the humidification means, wherein the controller startshumidification after a certain period of time has passed in accordancewith an instruction to mount the specimen.
 10. The culture microscopeapparatus according to claim 1, further comprising: a manipulator thathas a syringe, an arm capable of rotating and vertical movement tolocate the syringe on an optical axis position of the objective lens,and a drive portion to rotate and vertically move the arm.
 11. A culturemicroscope apparatus comprising: (i) a microscope device for observing aspecimen, the microscope device comprising: an objective lens forallowing the specimen to be observed, a moving device for relativelymoving the specimen and the objective lens, an image pickup device forpicking up an image of the specimen through the objective lens, an upperbase member on which the objective lens and the moving device areplaced, and a lower base member on which the image pickup device isplaced, the upper base member and the lower base member being both madeof a low expansion material, the upper base member and the lower basemember being connected by support columns; (ii) a culture device capableof controlling temperature and humidity, the culture device comprisingan isolation wall including an insulating material that thermallyisolates the image pickup device from the culture device; and (iii)isolation means for isolating the microscope device from moisture of theculture device.
 12. The culture microscope apparatus according to claim11, wherein the objective lens is a water-immersion objective lens, andthe microscope device further comprises a water supply device forsupplying water to the water-immersion objective lens, and the watersupply device comprises a cooler capable of setting a temperaturedifferent from a set temperature of the culture device, and a watermovement mechanism for moving water condensed in the cooler to thewater-immersion objective lens.
 13. A culture microscope apparatuscomprising: a microscope device for observing a specimen, wherein themicroscope device comprises illumination means for applying excitationlight to the specimen, specimen observing means for observing lightgenerated from the specimen due to the excitation light, and dimmermeans for dimming the excitation light that has penetrated the specimen;a culture device capable of controlling temperature and humidity; andisolation means for isolating the microscope device from moisture of theculture device.
 14. The culture microscope apparatus according to claim13, wherein a plurality of specimens are arranged in line, the specimenobserving means and the dimmer means are located to face each other witha space, the specimens are located between the specimen observing meansand the dimmer means, and the specimen observing means and the dimmermeans are movable along the line of the specimens.
 15. The culturemicroscope apparatus according to claim 13, wherein the dimmer meanscomprises a light absorption member which absorbs the excitation light.16. The culture microscope apparatus according to claim 13, furthercomprising an illumination light source for transmitted-lightillumination of the specimen, wherein light from the illumination lightsource can be applied to the specimen from an opposite side of thespecimen observing means.